Stable pharmaceutical composition of clopidogrel free base for oral and parenteral delivery

ABSTRACT

The present disclosure provides ready-to-use, oil/water emulsion compositions with mean droplet size (intensity-average, nm) of 100-500 nm, where the oil phase comprises clopidogrel free base dispersed in pharmaceutical acceptable oil(s). The emulsion uses clopidogrel free base or premix of clopidogrel free base in oil(s) as the starting materials and may also contain one or more excipients such as surfactant and or co-surfactant, osmotic agent, pH adjustment agent, antioxidant, preservative, sweetener, and/or suspending agent, etc.

PRIORITY CLAIM

This application claims priority to U.S. application Ser. No. 15/279,848filed on Sep. 29, 2016, which is a continuation of U.S. application Ser.No. 14/174,351 (now U.S. Pat. No. 9,480,680) filed on Feb. 6, 2014,which claims priority to U.S. Provisional Patent No. 61/761,234 filed onFeb. 6, 2013, the contents of all of which are hereby fully incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to oil/water emulsion compositionscontaining clopidogrel free base dispersed in oils that significantlyimprove the stability of clopidogrel against chiral, hydrolytic, andthermal degradations, and to the methods of making and using thereof inthe treatment of mammal, particularly human subjects in need ofclopidogrel.

BACKGROUND OF THE INVENTION

Clopidogrel, methyl(+)-(S)-α-(o-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-acetate,is an anticoagulant drug that inhibits platelet aggregation by selectivebinding to adenylate cyclase-coupled ADP receptors on the plateletsurface. It is widely used for the prevention of atherothrombotic eventssuch as myocardial infarction, stroke, peripheral arterial disease,acute coronary syndrome, and cardio-vascular death. The structure of theS-enantiomer of clopidogrel is shown below in structure (I):

Clopidogrel (Such as Plavix® and other generics) is currently onlyavailable as a tablet form containing 75 mg equivalents of theclopidogrel base; the drug is present in the bisulfate salt form. Thereis no liquid injectable or liquid oral dosage form currently availablein the market. PLAVIX® is an antiplatelet medication approved by theU.S. Food and Drug Administration to reduce thrombotic events andpatients with acute coronary syndrome. For an average loading dose (300mg clopidogrel in a PLA VIX® tablet), the typical time to reach thedesired therapeutic effect (e.g. platelet aggregation inhibition) variesfrom two to five hours probably due to delay in absorption, delay insystem availability, or suboptimal bioavailability. If there is animmediate need for the procedure (such as precutaneous coronaryintervention (PCI) in less than two to three hours), a larger thanaverage dose of clopidogrel is usually administered to patient in orderto achieve faster onset, which could potential cause deadly side effectssuch as hemorrhage, and long bleeding.

Therefore, there is an urgent need for liquid clopidogrel dosage formthat could be administered as injectable or oral solution to achieverapid therapeutic effect without increasing the dose. The ability toformulate clopidogrel in a biocompatible vehicle having minimum sideeffects and appropriate pharmadynamic profiles is critical to the use ofclopidogrel as an intravenous or oral liquid agent. This is particularchallenge for clopidogrel because of its lipophilicity, pH-dependentsolubility with very low solubility at physiological pH, and extremelychemical instability at alkaline pH.

Clopidogrel is a weakly base with pKa of 4.5. It is practicallyinsoluble in water at neutral pH but freely soluble at pH 1. It alsodissolves freely in methanol, dissolves sparingly in methylene chloride,and is practically insoluble in ethyl ether. It has a specific opticalrotation of about +56°. Clopidogrel free base is a semi-solid, oily formwith high-viscosity, which causes problems in storage or handlingprocess. Moreover, it has been reported that clopidogrel free base wasnot suitable for use in pharmaceutical dosage forms, which is unstableunder increased moisture and temperature. Due to a labile proton in thechiral center and the methyl ester group, it was very susceptible toracemization, oxidation, and hydrolysis of a methyl ester group. It wasreported that antioxidants did not prevent this degradation, and higherpH accelerated instability. As a result, it was indicated thatclopidogrel should only be stabilized with salt-forming acids beforeincorporated into dosage forms.

Clopidogrel bisulfate, which is currently used in the commercial oraltablet Plavix® (Sanofi Aventis), is one of the salt examples used inoral dosage form. It is supplied in tablet form containing 75 mgequivalents of the clopidogrel base. Similarly to free base, clopidogrelbiosulfate is also relatively unstable under increased moisture andtemperature and in alkaline pHs due to its susceptibility toracemization, oxidation, and hydrolysis of a methyl ester group.Clopidogrel is a chiral molecule and can therefore exist as an R or an Senantiomer. The S-enantiomer is biologically active, while theR-enantiomer (impurity C) does not exhibit any anti-aggregation activityand is poorly tolerated. It can evoke convulsions at high doses ofanimals. The major circulating compound after administration is theinactive carboxylic acid derivative, which is formed by hydrolysis ofthe ester function by carboxylesterase. Carboxylic acid derivative(S)-(+)-(2-chlorophenyl)-6,7-dihydro-4H-thieno[3,2-c]pyridine-5-ylacetic acid (clopidogrel acid, Impurity A), which can be obtained by thehydrolysis of the ester group, either in vitro, catalyzed by theincreased humidity, pH, and temperature, or in vivo, as a result of theaction of enzyme carboxylesterase, is the main degradation producthaving no pharmacological activity. This implies that the content of theR-enantiomer as well as the inactive carboxylic acid derivative must becarefully controlled in clopidogrel bulk substance and drug products.The structure of those impurities listed in USP 32 and EP are shownbelow:

The low solubility of clopidogrel in water at neutral pH makes it verydifficult to develop a bioavailable and physical stable pharmaceuticalproduct, particularly when intravenous or oral solutions are needed.Whereas its highly pH-dependent solubility make it very challenging tomake a suitable aqueous-based injectable dosage forms that will notprecipitate out when contact with body fluid and will not causeinjection pain, phlebitis, and even embolism upon administration.Moreover, chemical instability of clopidogrel, in the presence ofmoisture, heat, and alkaline pH, precludes the use of aqueous solvent inthe formulation, limits its formulation to organic solvent-based liquidor freeze-dried solid, and restrict its storage condition to a lowstorage temperature such as refrigeration or freezing.

A number of approaches for preparing intravenous and oral liquidcompositions of sparingly or poorly water-soluble basic drugs areavailable. These methods include micellar solubilization or drugnanoparticle suspension by means of surface-active agents; formation ofcomplexes with cyclodextrin and its derivatives (Hydroxypropylbeta-Cyclodextrin (HPBCD) and sulfobutylether-β-cyclodextrin (SBECD));use of various co-solvent systems; and formation of salt with strongacid with a low solution pH. However, for micellar system, surfactantshave been implicated by adverse effects such as hemolysis and histaminereaction and severe anaphylaxis reaction, and for nanosuspension systemwherein pure drug particles of nanosize stabilized by polymer andsurfactants, potential catalytic degradation of drug substance due tohigher exposure area to aqueous media and the surrounding surfactantshas been reported; taste masking and injection pain is another issue forthe micellar/nanosuspension system due to a higher concentration of freedrug available in the aqueous medium; co-solvent systems is known forcausing precipitation, injection pain and phlebitis; potentialnephrotoxicity and bradycardia and reduction of blood pressure caused bycyclodextrine and its derivitives and the potential concerns ofcyclodextrin binding with coadminstered lipophilic drugs have beenreported; and the low solution pH of weakly basic salt formed withstrong acid such as clopidogrel bisulfate will cause drug-excipient andproduct stability issue and cause tasting issue (clopidogrel has abittering taste), injection site irritation and pain as a result ofprecipitation of the drug as free base when contact with blood atneutral pH. In summary, each of these methods listed above has itsinherent limitations and are insufficient to formulate clopidogrel in abiocompatible vehicle having sufficient stability, minimum side effects,and appropriate pharmadynamic profiles as a either intravenous agent ororal solution agent.

WO 2008/060934 discloses an emulsion composition containing micronizedoil droplets of pure tetrahydropyridine anti platelet agent stabilizedby surfactant. Even though the publication mentions that the agent couldbe clopidogrel, the publication only discloses emulsion compositionsusing clopdogrel bisulfite as the starting material. Emulsioncomposition using clopidogrel free base as a starting material was notdisclosed in the publication, and effects of drug form (free base orsalt form) on the product stability, particularly on the drug-relatedimpurity profiles of clopidogrel was not disclosed for the compositioneither.

CN102697724 discloses oil in water emulsion made with clopidogrelbisulfate or amino acid salts as the starting materials. However, thepublication does not disclose emulsion compositions using clopidogrelfree base as the starting material and the concentration of clopidogrelfree base achieved in the emulsion composition is low (<0.15% w/v),which may require a large volume of emulsion to achieve the requireddose of 300 mg, In addition effects of drug form (free base or saltform) on the product stability, particularly on the drug-relatedimpurity profiles of clopidogrel was not disclosed for the compositionseither

SUMMARY OF THE INVENTION

To address the above mentioned flaws and problems in the current art,there is a need in the art for a clopidogrel liquid dosage form,particularly injectable, which is stable and ready-to-use, and canprovide a single IV infusion containing up to 300 mg of clopidogrel dosefor rapid therapeutic action, and can be prepared and stored at roomtemperature or refrigerated temperature. This disclosure provides suchcompositions. The compositions can be provided as anti-platelet agentfor patient under emergence and intense care or who cannot take oraltablet administration, with such stability and purity to meet therequirements of the applicable compendium, Food and Drug Administrationand GMP for use in preparation of a pharmaceutical formulation.

Considering the aforementioned problems, the present disclosure providesa surprising result that ready-to-use, aqueous-based oil/waterparenteral or oral emulsion compositions containing clopidogrel freebase with mean droplet size (intensity-average, nm) of 100-500 nm,wherein the oil phase comprises the clopidogrel free base dispersed inoils within acceptable limit of a surfactant and/or a co-surfactant,unexpectedly gives extremely favorable product stability and favorableimpurity profiles at the time of exposure to high pH of ˜9-10, or highpressure sterilization by water or steam, or after a long term storage,and thus superior quality and efficacy.

The present invention describes methods of controlling the amount ofsuch impurities in a pharmaceutical compositions having clopidogrel asan active ingredient that meet USP 32 impurity specification forclopidogrel tablet. The emulsion formulations significantly improved thestability of clopidogrel over other aqueous based formulations (such ascyclodextrin-based formulations) or emulsion made with clopidogrel salt(such as bisulfate salt) with respect to chiral, hydrolysis, and thermaldegradations.

-   -   1. One aspect of this invention is that the aqueous-based        emulsion of this invention significantly reduced the hydrolysis        of the methyl ester group (impurity A); Even though clopidogrel        itself is very unstable at alkaline pH, the emulsion formulation        prepared with pH adjusted to high pH at ˜9-10 surprisingly show        minimum amount of hydrolysis (<1.2%) over the shelf life,        whereas unformulated clopidogrel degraded completely in 5        minutes in diluted NaOH solution under the same condition.    -   2. Another aspect of the invention is that the emulsion        formulation of this invention surprisingly prevented thermal        degradation of clopidogrel. It was known from literature that        clopidogrel is unstable due to a labile proton in the chiral        center and was susceptible to oxidation and even antioxidants        did not prevent this degradation. To the contrary of the        literature, the emulsion formulation of this invention inhibit        the thermal degradation of clopidogrel without the use of        antioxidant.    -   3. Another aspect of the invention is that the emulsion of this        invention surprisingly inhibit chiral conversion of clopidogrel        from the S-enantiomer (biologically active) to the R-enantiomer        (which does not exhibit any anti-aggregation activity and is        poorly tolerated) (impurity C<1.5%). This is contrary to the        cyclodextrin-based solution formulation or emulsion formulation        using clopidogrel salt (such as bisulfate salt) as the starting        materials wherein significant amount of R-enantiomer was formed        during process and storage.    -   4. Another aspect of the present invention describes        pharmaceutical compositions prepared or stored using the        manufacturing process methods described herein wherein the level        of certain drug-related impurities is minimized or reduced.        -   a) In particular, the present invention describes            pharmaceutical composition having clopidogrel as an active            ingredient, and having a reduced level of impurities that            meet requirement of USP 32 impurity specifications for            clopidogrel tablet.        -   b) More specifically, the present invention describes a            pharmaceutical composition having clopidogrel as an active            ingredient, wherein the compositions contains equal or no            more than 1.2% of clopidogrel related compound A, not more            than 1.5% of clopidogrel related compound C, not more than            0.2% of any other single drug-related impurity (excluding            clopidogrel related compound B), and not more than 2.5% of            total impurities (excluding clopidogrel related compound B).    -   5. Another aspect of the present invention is a method of        treating or alleviating a disease or condition in a subject in        need thereof, comprising administering to the subject an        effective amount of a pharmaceutical composition having        clopidogrel free base as the active ingredient, wherein the        level of impurities is reduced or minimized to no more than 2.5%        total drug related impurity to clopidogrel (excluding        clopidogrel related compound B). As used herein the disease or        condition refers to any disease or condition which may be        treated using clopidogrel containing medicine for the prevention        of atherothrombotic events such as myocardial infarction,        stroke, peripheral arterial disease, acute coronary syndrome,        and cardio-vascular death.

It is an object of this invention to provide a stable pharmaceuticaloil/water emulsion composition with nanosized oil droplets forparenteral or oral administration omprising: clopidogrel free basedispersed in an oil phase; a surfactant and an optional a co-surfactant;a water phase substantially free of clopidogrel; and a pH adjustingagent.

It is another object of this invention to provide a method to make astable pharmaceutical oil/water emulsion composition with nanosized oildroplets for parenteral or oral administration, said method comprisingthe steps of: a) preparing an oil phase comprising clopidogrel bydispersing clopidogrel free base in an oil carrier; b) preparing anaqueous phase comprising water and a pH adjustment agent; c)incorporating a surfactant or optionally a co-surfactant in the oilphase or in the aqueous phase; d) dispersing the oil phase in theaqueous phase to form a coarse emulsion and adjusting pH to about 9; e)forming a final emulsion by sonicating or high pressure homogenizing theemulsion of step d), and adjusting pH to between 5.5 and 10; f)filtrating the final emulsion; and g) controlling product bioburden orsterility by aseptic process or terminal sterilization.

It is yet another object of this invention to a method to treat apatient in need of a single highdose of clopidogrel, said methodcomprising: a) providing in a liquid form of a pharmaceutical oil/wateremulsion composition prepared by dispersing clopidogrel free base in anoil carrier; preparing an aqueous phase comprising water and an pHadjustment agent; dispersing the oil phase into the aqueous phase bysonicating or homogenizing to form nanosized oil droplets; and b)administering orally or parenterally a single dose of the composition,wherein the single dose contains up to 300 mg of clopidogrel free base.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the mean droplet size (intensity-average, nm) of theemulsions prepared with clopidogrel free base (Example 10) andclopidogrel bisulfate (Example 8) after autoclave and freeze-thawconditions and storage under refrigeration (at ˜5° C.) for 1 year.

FIG. 2 HPLC Chromatogram of emulsion of clopidogrel free base afterstorage under refrigeration (at ˜5° C.) for 1 year (Example 10).

FIG. 3 shows comparison of percentage of clopidogrel chiral conversationfrom S to R stored at 40 degree Celsius between the clopidogrel emulsionof this invention (Example 10) vs clopidogrel-HPBCD and SBECD complex atpH of approximately 8.

FIG. 4 shows comparison of percentage of clopidogrel chiral conversationform S to R when stored at 25 degree Celsius between the clopidogrelemulsion of this invention (Example 10) vs clopidogrel-HPBCD and SBECDcomplex at pH of approximately 8.

FIG. 5 shows comparison of percentage of clopidogrel chiral conversationform S to R when stored at 40 degree Celsius between emulsion preparedusing clopidogrel free base (Example 10) vs emulsion prepared withclopidogrel bisulfate salt (Example 8).

FIG. 6. Overlay of emulsion (Example 6) droplet size distribution forfreshly prepared emulsion sample, emulsion sample after 19 weeks storageat room temperature, and emulsion sample after 19 weeks storage at 40°C.

DETAILED DESCRIPTION OF THE INVENTION

The term “Clopidogrel drug substance” or “clopidogrel free base” isdefined as: [methyl(+)-(S)-α-(o-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-acetate].

The term “clopidogrel related compound A” or “Impurity A” is defined as:[(+)-(S)-(o-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-aceticacid].

The term “clopidogrel related compound B” or “Impurity B” is defined as:[methyl(±)-(o-chlorophenyl)-4,5-dihydrothieno[2,3-c]pyridine-6(7H)-acetate].

The term “clopidogrel related compound C” or “Impurity C” is defined as:[methyl0-(R)-(o-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-acetate].

The invention provides aqueous-based oil in water emulsion formulationcomposition with mean droplet size (intensity-average, nm) of 100-500nm, comprising clopidogrel free base dispersed in oil(s) and asurfactant and/or a co-surfactant, and an aqueous liquid carrier. Theformulation of the composition of this invention:

a) clopidogrel free base,b) an oil phase,c) a surfactant, and/or a co-surfactant, andd) water and pH adjustment agent.

Optionally, the emulsion formulation may also contain chelate agent,antioxidant, osmotic agent, preservative, and buffering agent.

In some embodiments, the formulation further comprises a solubilizingagent, a flavoring agent, a sweetening agent, a viscosity inducingagent, electrolyte, another therapeutic agent, or a combination thereof.

Combinations of the various upper and lower limits to clopidogrel andother composition, as set forth in this disclosure, can be used toprovide different embodiments of the invention. The invention alsoprovides a method of administering clopidogrel comprising administeringa ready-to-use liquid dosage formulation comprising clopidogrel freebase dissolved in oil phase and aqueous phase thereof.

Emulsions of the invention offer much better stability and/or less sideeffects than other organic based or aqueous based dosage forms such ascyclodextrin-based formulation. Oil-in-water emulsions also prevent thelipophilic clopidogrel from adherence to the plastic infusion sets etc.during administration. The present invention also provides an emulsionof clopidogrel that is pharmaceutically stable at room temperature orrefrigerated temperature and that does not require dilution prior toadministration. Moreover, the emulsions give a fast release, togetherwith faster therapeutic effect than conventional oral tablet dosageforms.

In some embodiments, the formulation does not require dilution prior toadministration to a subject. In other embodiments; the liquidformulation may be diluted without precipitation of the clopidogrel. Theformulation of the invention can be a single-dose or multi-doseformulation.

Some embodiments of the methods of the invention include those whereinthe liquid formulation is administered by intravenous, oral, or entericroute. The present invention also provides a method of treating,preventing or reducing the occurrence of a disease associated withplatelet aggregation or of a disease that requires clopidogrel therapy,the method comprising administering the formulation of the invention toa subject in need thereof. The invention also provides a method ofdecreasing the time to therapeutic onset or the time required to reachthe target therapeutic effect provided by clopidogrel, comprisingadministering by parenteral, enteric, or oral route to a subject in needthereof, a formulation according to the invention. The formulation ofthe invention could provide a reduced time to therapeutic onset and/orto target therapeutic effect as compared to an oral solid tablet dosageform. The formulation of the invention also permits administration of alower dose of clopidogrel to achieve a target therapeutic effect, e.g.target bleeding time or target inhibition of platelet aggregation, ascompared to administration of a reference solid tablet oral dosage formto achieve the same target therapeutic effect.

According to one embodiment of this invention the emulsion comprises:

a) 0.01-10% w/w of clopidogrel free base;b) 1-30% w/w of oil phase;c) surfactant of 0.5-5.4% w/w;d) co-surfactant of 0-0.5% (optional); andd) 60-99% w/w of water and pH adjustment agent, such as sodium hydroxideto adjust pH above 5.

The Clopidogrel free base used for the formulation is a chiral moleculeand can therefore exist as S-enantiomer. The S-enantiomer isbiologically active, while the R-enantiomer (impurity C) does notexhibit any anti-aggregation activity and is poorly tolerated.

According USP 32, the active pharmaceutical ingredient used in thecommercial tablet product, i.e. clopidogrel bio sulfate is mainlyS-enantiomer, i.e. not more than 0.2% of clopidogrel related compound Anot more than 0.3% of the first enantiomer of clopidogrel relatedcompound B; not more than 1.0% of clopidogrel related compound C; notmore than 0.1% of any other drug-related impurity is found; and not morethan 1.5% of total drug-related impurities.

As for the finished product after preparation and during storage, thecommercial tablet product contains not more than 1.2% of clopidogrelrelated compound A, not more than 1.5% of clopidogrel related compoundC, not more than 0.2% of any other single drug-related impurity(excluding clopidogrel related compound B), and not more than 2.5% oftotal drug-related impurities (excluding clopidogrel related compound B)according to USP32.

It has been generally recognized that clopidogrel free base was notsuitable for use in pharmaceutical dosage forms due to its instabilityunder increased moisture and temperature and susceptibility toracemization, oxidation, and hydrolysis of its methyl ester group, andhat clopidogrel should only be stabilized with salt-forming acids beforeincorporated in to dosage forms. In this invention, use of clopidogrelfree base is preferred over clopidogrel salt. Due to low solubility ofclopidogrel salt in pharmaceutical-acceptable oil(s), its partitionbetween oil and aqueous phase is low. As a result, the drugencapsulation efficient of clopidogrel dissolved in the oil phase (drugloading in oil phase) is low for clopidogrel salt. Therefore,formulation using clopidogrel salt as the drug substance will not besuitable to make emulsion containing a high drug loading (>0.15%clopidogrel free base drug load) when administration of clopidogreldosage form as a single dose in a reasonable dose volume (such as 300 mgin 100 ml or less) is desired, unless very high level of surfactants orsolvent are used. Otherwise, clopidogrel may crash out of the oil phase,dissolve, or precipitate into the aqueous phase, which will causeproduct homogeneity problem (presence of two forms, one is clopidogreldissolved in oil, the other is free clopidogrel suspended in aqueousmedium), stability problems as a result of clopidogrel exposed directlyto water and ions that catalyze the degradation reaction, and injectionsite irritation/pain for injection and taste masking issue for oraldosage, and physical instability (precipitation) upon IV administration.In one embodiment of this invention, clopidogrel free base is used as isobtained from suppliers; in another embodiment, a composite containingthe free base dissolved in pharmaceutical acceptable oil(s) is used asthe starting materials for formulation. In another embodiment,clopidogrel free base is obtained by conversion of a clopidogrel salt tofree base and separation of the counter ion from the free base prior tomanufacturing.

The preferred range of clopidogrel free base in the formulation is0.15-10%. The most preferred range of clopidogrel free base in theformulation is 0.2-3%.

Oil phases in the emulsion are any pharmaceutical-grade oil, preferablytriglycerides such as, but not limited to soy bean oil, safflower seedoil, olive oil, cottonseed oil, sunflower oil, fish oil (containing theomega-3 fatty acids eicosapentaenoic acid (EPA), and docosahexaenoicacid (DHA)), castor oil, sesame oil, peanut oil, corn oil, and mediumchain triglycerides (such as Miglyol 812 or 810). The oil phase may alsocontain surfactant and/or co-surfactant such as egg lecithin, soylecithin, and other phosphorus lipids, propylene glycol diesters, oleicacid, or monoglycerides (such as acetylareal monoglycerides). The oilphase can also be a mixture of said ingredients.

The preferred lipid phase is soy bean oil, medium chain triglycerides(MCT), olive oil, and fish oil, either alone or mixture with others.

The most preferred oil phase is soy bean oil. The preferred range of oilcarrier is 5-30%. The most preferred rang of oil carrier is 10-20%.

Surfactants are any pharmaceutically acceptable surfactant, preferablyphospholipids extracted from egg yolk or soy bean, syntheticphosphatidyl cholines or purified phosphatidyl cholines from vegetableorigin. Hydrogenated derivatives can also be used, such as phosphatidylcholine hydrogenated (egg) and phosphatidyl choline hydrogenated (soy).Surfactants may also be non-ionic surfactants such as poloxamers (forexample Poloxamer 188 and 407), poloxamines, polyoxyethylene stearates,polyoxyethylene sorbitan fatty acid esters or sorbitan fatty acidesters. Ionic suffactants may also be used such as cholic acid anddeoxycholic acid or surface active deriviatives or salts thereof.

The most preferred surfactant is egg lecithin. The preferred range ofsurfactant is 0.6-2.4%. The most preferred range of surfactant is1.2-1.8%.

The co-surfactant is selected from the group consisting of oleic acid,sodium oleate, cholic acid, sodium cholate, deoxycholic acid,deoxysodium cholate and a mixture thereof; wherein the saidco-surfactant is presented in the said invention in the range of 0-0.5w/v % of the composition.

The preferred range of water of buffer is 70-90%.

The emulsion may also contain co-solvents or other solubility enhancers,chelate agent, preservative, antioxidants, stabilizers, pH-adjustingagents or tonicity modifying agents, such as glycerol, polymer assuspending agent, and sweetener, etc.

Desirable emulsions are stable systems of intensity-average droplet sizeof 100-1000 nanometer with white to off-white color. The preferredintensity-average droplet size is 100-500 nanometer; the most preferredintensity-average droplet size is 100-300 nanometer.

The preferred pH range of the emulsion after manufacturing and duringstorage is 5.5 and above. In one embodiment, the pH of the emulsion iscontrolled to the range to 5.5-7; in another embodiment, the pH of theemulsion was controlled to 7-10. The preferred pH range of the emulsionis 6.5-9. The pH adjustment agent can be a buffer or sodium hydroxide orother pH adjustment agents or combine thereof.

The emulsion of the invention can be prepared in the following method:For the aqueous phase, pharmaceutical-grade water is dispensed to acontainer and heated to about 40-80° C. Egg lecithin and glycerin isadded and pH is adjusted to 9-10. For the oil phase, soybean oil isdispensed into another container and heated to about 40-80° C.Clopidogrel and optionally co-surfactant is then added to the soybeanoil and heated to about 40° C. to about 80° C. Optionally, egg lecithincan be added to the oil phase. The aqueous and oil phases are then mixedtogether by a high shear mixer to form a coarse emulsion. The emulsionis then sonicated or homogenized with a high pressure homogenizer or amicro-fluidizer at a pressure of about 5000-15000 psi and a temperaturerange of about 5° C. to about 60° C. to obtain an emulsion with adesired droplet size. The pH is adjusted with pH adjustment agent suchas 1 N sodium hydroxide solution to a pH of about 5.5 to 10. In oneembodiment, pH is adjusted to 9-10. In another embodiment, pH isadjusted to 7-10. In another embodiment, pH is adjusted to 8-10. Thesamples are filtered and dispensed into cleaned bottles, often withnitrogen gas overlay, and capped with siliconized rubber stoppers, andcrimp sealed with an aluminum seal. The product can be manufactured byan aseptic process or by terminal sterilization. Preferably the dosageunits are autoclaved to get sterile and stable emulsions. In oneembodiment, the emulsion was autoclaved at 121° C. for 15-20 minutes. Inanother embodiment, the emulsion is processed aseptically under sterileenvironment without autoclave.

The invention is now described by way of non limiting examples. Theinvention comprises combinations of the embodiments and aspects of theinvention as detailed herein. Accordingly, the invention also includescombinations and sub-combinations of the individual elements of theembodiments or aspects of the invention as described herein. Otherfeatures, advantages and embodiments of the invention will becomeapparent to those skilled in the art by the following description,accompanying examples. The disclosure herein is directed to all suchvariations and modifications to such elements and methods known to thoseskilled in the art. Furthermore, the embodiments identified andillustrated herein are for exemplary purposes only, and are not meant tobe exclusive or limited in their description of the present invention. Askilled artisan would realize that various changes and modifications maybe made without diverting from the spirit of the invention.

Example 1. Comparison of Drug Partition Between Oil and Aqueous Phasefor Clopidogrel Bisulfate and Free Base

In order to determine percentage of clopidogrel partitioning in oil andaqueous phases, a partition study was conducted using clopidogrelbiosulfate and clopidogrel free base. 600 mg of drug was weighed out andadded to a beak containing equal weight of soy bean oil and DI water (20g each). Stir the mixture in room temperature for 24 hours. At the endof study, samples from both phases were withdrawn and tested by HPLC asdescribed in Example 14. The pH of the aqueous phase were measured.

Table 1 shows that that there could be about 50% of clopidogrel stayingin the aqueous phase when clopidogrel bisulfate is used for the study.To the contrary, the amount of clopidogrel in the aqueous phase isnegligible when free base is used for the partition study. This studysuggests that if we would like to minimize amount of free clopidogrel inthe aqueous phase, the free base will be preferred over its bisulfatesalt. Otherwise, there could have formulation inhomogeneity andstability problems.

TABLE 1 Oil/water partition study Concentration (mg/mL) mg/mL PhaseBisulfate salt Free base Oil Phase 17 29 Aqueous Phase 15 0.006 O/Wpartition ratio 1.1 4833 pH of aqueous phase 2 7

Example 2. Preparation of Emulsion Using Clopidogrel Free Base Obtainedas is from the Supplier

Quantities: Formula g Clopidogrel free base 0.20 soy bean oil 10.0 egglecithin 1.2 glycerol 2.25 sodium hydroxide q.s. to pH 9-10 Water forInjections to 100 g

All processing stages are carried out under nitrogen.

A sterile aqueous oil-in-water emulsion for parenteral administration isprepared as follows:

-   -   1. An aqueous phase is prepared from glycerol, and Water for        Injections. The pH of the aqueous phase is adjusted to ˜9-10        with 1 N sodium hydroxide solution. This mixture is stirred and        heated to a temperature of approximately 60° C.    -   2. The aqueous phase is passed through a 0.22 micron filter and        charged to a mixing vessel.    -   3. Separately, an oil phase is prepared from soy bean oil that        has been passed through a 0.22 micron filter, clopidogrel free        base and egg lecithin, in a vessel. The mixture is stirred at a        temperature of approximately 60° C. until all ingredients are        dissolved.    -   4. The mixture is added to the aqueous phase.    -   5. This mixture is then mixed with a high shear mixer (Polytron        PT3100) at 10,000 rpm for 5 minutes to obtain a coarse emulsion.        The emulsion pH is adjusted to 9-10.    -   6. The mixture is then sonicated with an ultrsonic processor        (Fisher Scientific Sonic Dismembrator, Model 500) with for 30        minutes. The product temperature is controlled at ˜45 degree C.    -   7. The resultant oil-in-water emulsion is cooled, pH adjust to        9-10 if necessary, and then transferred into a filling vessel.    -   8. The emulsion is then filtered with 0.45 micron filter and        filled into containers under nitrogen and autoclaved at 121 C        for 20 minutes.    -   9. The final pH is ˜8.

Example 3. Preparation of Emulsion Using Clopidogrel Free Base Obtainedas is from the Supplier

Quantities: Formula g Clopidogrel free base 0.6 soy bean oil 10.0 egglecithin 1.8 glycerol 2.25 sodium hydroxide q.s. to pH 9-10 Water forInjections to 100 g

All processing stages are carried out under nitrogen.

A sterile aqueous oil-in-water emulsion for parenteral administration isprepared as follows:

-   -   1. An aqueous phase is prepared from glycerol, lecithin and        Water for Injections. The pH of the aqueous phase is adjusted to        ˜9-10 with 1 N sodium hydroxide solution. This mixture is        stirred and heated to a temperature of approximately 60° C.    -   2. The aqueous phase is passed through a 0.22 micron filter and        charged to a mixing vessel.    -   3. Separately, an oil phase is prepared from soy bean oil that        has been passed through a 0.22 micron filter, and clopidogrel        free base in a vessel. The mixture is stirred at a temperature        of approximately 60° C. until all ingredients are dissolved.    -   4. The mixture is added to the aqueous phase.    -   5. This mixture is then mixed with a high shear mixer (Polytron        PT3100) at 10,000 rpm for 5 minutes to obtain a coarse emulsion.        The emulsion pH is adjusted to 9-10.    -   6. The mixture is then homogenized with a high pressure        homogenizer (APV 2000) at ˜10,000 psi for 10 cycles. The product        temperature is controlled at ˜45 degree C.    -   7. The resultant oil-in-water emulsion is cooled, pH adjust to        9-10 if necessary, and then transferred into a filling vessel.    -   8. The emulsion is then filtered with 0.45 micron filter and        filled into containers under nitrogen and autoclaved at 121 C        for 20 minutes.    -   9. The final pH is ˜8.

Example 4. Preparation of Emulsion Using Clopidogrel Free Base Obtainedas is from the Supplier

Quantities: Formula g Clopidogrel free base 3.0 soy bean oil 10.0 egglecithin 1.2 glycerol 2.25 sodium hydroxide q.s. to pH 9-10 Water forInjections to 100 g

All processing stages are carried out under nitrogen.

A sterile aqueous oil-in-water emulsion for parenteral administration isprepared as follows:

-   -   1. An aqueous phase is prepared from glycerol and Water for        Injections. The pH of the aqueous phase is adjusted to ˜9-10        with 1 N sodium hydroxide solution. This mixture is stirred and        heated to a temperature of approximately 60° C.    -   2. The aqueous phase is passed through a 0.22 micron filter and        charged to a mixing vessel.    -   3. Separately, an oil phase is prepared from soy bean oil that        has been passed through a 0.22 micron filter, lecithin, and        clopidogrel free base in a vessel. The mixture is stirred at a        temperature of approximately 60° C. until all ingredients are        dissolved.    -   4. The oil mixture is gradually added to the aqueous phase under        high shear mixing.    -   5. This mixture is then mixed with a high shear mixer (Polytron        PT3100) at 10,000 rpm for 5 minutes to obtain a coarse emulsion.        The emulsion pH is adjusted to 9-10.    -   6. The mixture is then homogenized with a high pressure        homogenizer (APV 2000) at 10,000 psi for 10 cycles. The product        temperature is controlled at ˜45 degree C.    -   7. The resultant oil-in-water emulsion is cooled, pH adjust to        9-10 if necessary, and then transferred into a filling vessel.    -   8. The emulsion is then filtered with 0.45 micron filter and        filled into containers under nitrogen and autoclaved at 121 C        for 20 minutes.    -   9. The final pH is ˜8.

Example 5. Preparation of Emulsion Using Clopidogrel Free Base Obtainedas is from the Supplier by Aseptic Process

Preparation of Emulsion Using Clopidogrel Free Base Obtained as is fromthe Supplier

Quantities: Formula g Clopidogrel free base 3.0 soy bean oil 10.0 egglecithin 1.8 glycerol 2.25 Vitamin E 0.06 sodium hydroxide q.s. to pH8-10 Water for Injections to 100 g

All processing stages are carried out under nitrogen.

A sterile aqueous oil-in-water emulsion for parenteral administration isprepared as follows:

-   -   1. An aqueous phase is prepared from glycerol and Water for        Injections. The pH of the aqueous phase is adjusted to ˜9-10        with 1 N sodium hydroxide solution. This mixture is stirred and        heated to a temperature of approximately 60° C.    -   2. The aqueous phase is passed through a 0.22 micron filter and        charged to a mixing vessel.    -   3. Separately, an oil phase is prepared from soy bean oil that        has been passed through a 0.22 micron filter, lecithin, and        clopidogrel free base in a vessel. The mixture is stirred at a        temperature of approximately 60° C. until all ingredients are        dissolved.    -   4. The oil phase is then added to the aqueous phase.    -   5. This mixture is then mixed with a high shear mixer (Polytron        PT3100) at 10,000 rpm for 5 minutes to obtain a coarse emulsion.        The emulsion pH is adjusted to 9-10.    -   6. The mixture is then homogenized with a high pressure        homogenizer (APV 2000) at 10,000 psi for 10 cycles. The product        temperature is controlled at ˜45 degree C.    -   7. The resultant oil-in-water emulsion is cooled, pH adjust to        8-10 if necessary, and then transferred into a filling vessel.    -   8. The emulsion is then filtered with 0.45 micron filter and        filled into containers under nitrogen.    -   9. The final pH is ˜8.

Example 6. Preparation of Emulsion Using Clopidogrel Free Base Obtainedas is from the Supplier by Aseptic Process

Preparation of Emulsion Using Clopidogrel Free Base Obtained as is fromthe Supplier

Quantities: Formula g Clopidogrel free base 3.0 soy bean oil 10.0 egglecithin 1.2 glycerol 2.25 sodium hydroxide q.s. to pH 8-10 Water forInjections to 100 g

All processing stages are carried out under nitrogen.

A sterile aqueous oil-in-water emulsion for parenteral administration isprepared as follows:

1. An aqueous phase is prepared from glycerol and Water for Injections.The pH of the aqueous phase is adjusted to ˜9-10 with 1 N sodiumhydroxide solution. This mixture is stirred and heated to a temperatureof approximately 60° C.2. The aqueous phase is passed through a 0.22 micron filter and chargedto a mixing vessel.3. Separately, an oil phase is prepared from soy bean oil that has beenpassed through a 0.22 micron filter, lecithin, and clopidogrel free basein a vessel. The mixture is stirred at a temperature of approximately60° C. until all ingredients are dissolved.4. The oil phase is then added to the aqueous phase under high shearmixing.5. This mixture is then mixed with a high shear mixer (Polytron PT3100)at 6,000 rpm for 5 minutes to obtain a coarse emulsion. The emulsion pHis adjusted to 9-10.6. The mixture is then homogenized with a high pressure homogenizer (APV2000) at 10,000 psi for 10 cycles. The product temperature is controlledat ˜45 degree C.7. The resultant oil-in-water emulsion is cooled, pH adjust to 8-10 ifnecessary, and then transferred into a filling vessel.8. The emulsion is then filtered with 0.45 micron filter and filled intocontainers under nitrogen.9. The final pH is ˜8.

Example 7. Preparation of Emulsion Using Clopidogrel Free Base andLecithin as Surfactant And Oleic Acid as Co-Surfactant

Quantities: Formula g Clopidogrel free base 0.2 soy bean oil 10.0 egglecithin 1.2 Oleic acid 0.03 glycerol 2.25 sodium hydroxide q.s. to pH9-10 Water for Injections to 100

All processing stages are carried out under nitrogen.

A sterile aqueous oil-in-water emulsion for parenteral administration isprepared as follows:

-   -   1. An aqueous phase is prepared from glycerol, egg lecithin, and        Water for Injections. The pH of the aqueous phase is adjusted to        ˜9-10 with 1 N sodium hydroxide solution. This mixture is        stirred and heated to a temperature of approximately 60° C.    -   2. The aqueous phase is passed through a 0.22 micron filter and        charged to a mixing vessel.    -   3. Separately, an oil phase is prepared from soy bean oil that        has been passed through a 0.22 micron filter, oleic acid, and        clopidogrel free base in a vessel. The mixture is stirred at a        temperature of approximately 60° C. until all ingredients are        dissolved.    -   4. The oil mixture is added to the aqueous phase under high        shear mixing.    -   5. This mixture is then mixed with a high speed mixer        (Polytron 3100) at ˜10,000 rpm for 5 minutes to obtain a coarse        emulsion.    -   6. The mixture is then sonicated with an ultrsonic processor        (Fisher Scientific Sonic Dismembrator, Model 500) with for 30        minutes. The product temperature is controlled at ˜45 degree C.    -   7. The resultant oil-in-water emulsion is cooled, pH adjust to        9-10, and then transferred into a filling vessel.    -   8. The emulsion is then filtered and filled into containers        under nitrogen and autoclaved at 121 C for 20 minutes.    -   9. The final pH is ˜8.

Example 8. Preparation of Emulsion with Clopidogrel Biosulfate(Comparative Example)

Quantities: Formula g Clopidogrel bisulfate 0.26 (equivalent to 0.2 g offree base) soy bean oil 10.0 egg lecithin 1.2 glycerol 2.25 sodiumhydroxide q.s. to pH 9-10 Water for Injections to 100 g

Follow the procedure described in Example 2 without autoclave. The finalpH is ˜7.4

Example 9. Preparation of Emulsion Using Clopidogrel Free Base

Quantities: Formula g Clopidogrel free base 2.0 soy bean oil 10.0 egglecithin 1.2 glycerol 2.25 sodium hydroxide q.s. to pH 9-10 Water forInjections to 100

Follow the procedure described in Example 2. The final pH is ˜8.

Example 10. Preparation of Emulsion Using Premix of Clopidogrel FreeBase in Soy Bean Oil Obtained by Conversion of Clopidogrel BisulfateSalt to Free Base and Separation of the Sulfate Ion from the Free BasePrior to Manufacturing

Quantities: Formula g Premix of clopidogrel free base Equivalent to 0.20g of in soy bean oil free base soy bean oil QS ad. 10.0 egg lecithin1.2  glycerol 2.25 sodium hydroxide q.s. to pH 9-10 Water for Injectionsto 100 g

All processing stages are carried out under nitrogen.

Preparation of Premix of Clopidogrel Free Base in Soy Bean Oil

-   -   1. Dissolve clopidogrel bisulfate salt in sufficient amount of        water. Under continuous stirring, disperse required amount of        soy bean oil into the aqueous solution and add 1 N sodium        hydroxide by droplet wise to the mixture until the pH of the        aqueous phase is 6.5 and above. Separate the oil phase        containing the free base from the aqueous phase containing the        sulfate and sodium ions, and wash the oil phase with water if        necessary.

A sterile aqueous oil-in-water emulsion for parenteral administration isprepared as follows:

-   -   1. The drug-oil pre-mixture is heated under stirring to a        temperature of approximately 60° C. Then, the oil phase is        passed through a 0.22 micron filter and charged to a mixing        vessel.    -   2. An aqueous phase is prepared from glycerol, egg lecithin, and        water for Injections. The pH of the aqueous phase is adjusted to        ˜9-10 with 1 N sodium hydroxide solution. This mixture is        stirred and heated to a temperature of approximately 60° C.    -   3. The aqueous phase is passed through a 0.22 micron filter and        charged to a mixing vessel.    -   4. The oil mixture is added to the aqueous phase under high        shear mixing.    -   5. The mixture is then mixed with a high shear mixer (Polytron        PT3000) at ˜10,000 rpm for 5 minutes to obtain a coarse        emulsion.    -   6. The mixture is then circulated through a high pressure        homogeniser at 10,000 psi for 5 cycles. The product temperature        is controlled at ˜45 degree C.    -   7. The resultant oil-in-water emulsion is cooled, pH adjust to        ˜9-10, and then transferred into a filling vessel.    -   8. The emulsion is then filtered with 0.45 micron filter and        filled into containers under nitrogen and autoclaved at 121 C        for 20 minutes.    -   9. The final pH is ˜8.

Example 11. Preparation of Emulsion Using Clopidogrel Free Base

Quantities: Formula 2 g clopidogrel free base 0.20 g soy bean oil 10.0 gegg lecithin 1.8  glycerol 2.25 Vitamin E 0.06 sodium hydroxide q.s. topH 9-10 Water for Injections to 100 g 

Follow the procedure described in Example 3. The final pH is ˜8.

Example 12. Preparation of Emulsion Using Clopidogrel Free Base

Quantities: Formula 2 g clopidogrel free base   2.8 g soy bean oil  0.17g egg lecithin 0.54 glycerol 2.25 sodium hydroxide q.s. to pH 9-10 Waterfor Injections to 100 g

Follow the procedure described in Example 3. The final pH is ˜8.

Example 13. Characterization of Emulsion Droplet Size Distribution byNanoZeta Sizer: Comparison Emulsions Made with Clopidogrel Free Base andBisulfate Salt

The emulsion obtained from Example 8 and 10 were tested for particlesize distribution. Malvern Zetasizer Nano-ZS ZEN3600 was used to measurethe emulsion droplet size distribution. FIG. 1 shows the mean dropletsize (intensity-average, nm) of the emulsions prepared with clopidogrelfree base (Example 10) and clopidogrel bisulfate (Example 8). Theemulsion using free base is stable upon autoclave, freeze thaw andstorage under refrigeration for 1 year, whereas the emulsion usingclopidogrel bisulfate showed dramatic change in particle size afterautoclave and freeze thaw.

Example 14. Comparison Chemical Stability Between Emulsion of CurrentInvention and SB-E-CD and HP-B-CD Complex HPLC Method

A chiral LC method was used for impurity testing and enantiospecificassay. As stationary phase, an ULTRON ES-OVM column, 5 um (4.6 mm×150 mmi.d.) was used. The mobile phase consisted of Mobile Phases: A, 1.36 gSodium Phosphate monobasic (NaH2PO4.H2O) in 1.0 L purified water; B,acetonitrile. Binary gradient with constant flow rate of 1.0 mL/min and18 minute total run time. The injection volume 5-10 μl and UV detectionwas performed at 220 nm.

The major degradation impurities for clopidogrel emulsion of thisinvention were found to be impurity C (Chiral R). The hydrolyticdegradation impurity A was the minor degradant (<1.2%) for the emulsionproduct, and no significant change in the Impurity A was observed.Therefore, the chemical stability of the emulsion product of thisinvention was monitored with Impurity C (FIG. 2). The stability data(FIGS. 3 and 4) of emulsion using free base showed better stability thancyclodextrin-based clopidogrel solution.

FIG. 3 shows comparison of percentage of clopidogrel chiral conversationfrom S to R stored at 40 degree Celsius between the clopidogrel emulsionof this invention (Example 10) vs clopidogrel-HPBCD and SBECD complex atpH of approximately 8 (literature value from US20100292268).

FIG. 4 shows comparison of percentage of clopidogrel chiral conversationform S to R when stored at 25 degree Celsius between the clopidogrelemulsion of this invention (Example 10) vs clopidogrel-HPBCD and SBECDcomplex at pH of approximately 8 (literature value from US20100292268).

Example 15. Comparison of Emulsions Chemical Stability Made withClopidogrel Free Base (Example 10) and Bisulfate Salt (Example 8)

HPLC Method described in Example 14 was followed.

The stability data (FIG. 5) of emulsion using clopidogrel free baseshowed better stability than the one prepared with clopidogrel sulfatesalt.

FIG. 5 shows comparison of percentage of clopidogrel chiral conversationform S to R when stored at 40 degree Celsius between emulsion preparedusing free base (Example 10) vs emulsion prepared with clopidogrelsulfate salt (Example 8).

Example 16. Stability Summary of Emulsion of Example 10

Stability data in Table 2 below shows that the emulsion of example 10 isstable at least for 52 weeks when stored under refrigeration.

TABLE 2 Stability summary of Emulsion Example 10 Stability conditions(~5° C.) Weeks 0 12 14 20 52 Impurity C 0 0.56% 0.50% 1.04% 0.24%Droplet 198.4 NT NT NT 198.1 size(d50), nm pH 8.10 NT NT NT 6.18

Example 17. Stability Summary of Emulsion of Example 6

Stability data in Tables 3 and 4 below shows that the emulsion ofexample 6 is stable at least for 19 weeks when stored underrefrigeration (˜5° C.) or room temperature (˜25° C.).

TABLE 3 Stability summary of Example 6 Stability conditions at ~25° C.weeks 0 19 Impurity C 0.10% 0.90% Droplet size (D50), nm 191.7 207.6 pH7.8 6.6

TABLE 4 Stability summary of Example 6 Stability conditions at ~5° C.weeks 0 19 Impurity C 0.10% 0.14% Droplet size (D50), nm 192.0 193.0 pH7.8 6.7

What is claimed is:
 1. A method to make a stable pharmaceuticaloil/water emulsion composition with nanosized oil droplets forparenteral or oral administration, said method comprising the steps of:a) preparing an oil phase comprising clopidogrel by dispersingclopidogrel free base in an oil carrier; b) preparing an aqueous phasecomprising water and a pH adjustment agent; c) incorporating asurfactant or optionally a co-surfactant in the oil phase or in theaqueous phase; d) dispersing the oil phase in the aqueous phase to forma coarse emulsion and adjusting pH to about 9; e) forming a finalemulsion by sonicating or high pressure homogenizing the emulsion ofstep d), and adjusting pH to between 5.5 and 10; f) filtrating the finalemulsion; and g) controlling product bioburden or sterility by asepticprocess or terminal sterilization.
 2. The method of claim 1, whereinclopidogrel free base is obtained by converting clopidogrel salt to freebase and separating counter ion from the free base.
 3. The method ofclaim 1, wherein clopidogrel free base is provided as a free base-oilcarrier premix.
 4. The method of claim 1, wherein the oil carrier is soybean oil, fish oil, medium chain triglycerides, olive oil, or mixture ofthem thereof, and the surfactant is egg lecithin.
 5. The method of claim1, wherein the pH in step e) is adjusted to between 7 and
 10. 6. Amethod to treat a patient in need of a single highdose of clopidogrel,said method comprising: a) providing in a liquid form of apharmaceutical oil/water emulsion composition prepared by dispersingclopidogrel free base in an oil carrier; preparing an aqueous phasecomprising water and an pH adjustment agent; dispersing the oil phaseinto the aqueous phase by sonicating or homogenizing to form nanosizedoil droplets; and b) administering orally or parenterally a single doseof the composition, wherein the single dose contains up to 300 mg ofclopidogrel free base.
 7. The method of claim 6, wherein the single doseof the composition contains no more no more than 1.2% of impurity A, nomore than 1.5% of impurity C.
 8. The method of claim 6, wherein in stepa) pH of the composition is adjusted to between 7 and 10.